Detection of nucleic acids by type-specific hybrid capture methods

ABSTRACT

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid sequences. The method produces DNA/RNA hybrids which can be detected by a variety of methods.

This application is a Divisional of U.S. application Ser. No.12/622,160, filed Nov. 19, 2009, which is a Continuation of U.S.application Ser. No. 10/311,645, filed Apr. 4, 2003, which is a §371National Stage of PCT/US01/19353, filed Jun. 15, 2001, which is acontinuation-in-part of U.S. application Ser. No. 09/594,839, filed Jun.15, 2000, the contents of each of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to the field of nucleic acid detection methods ingeneral and more particularly relates to the detection of nucleic acidsby target-specific hybrid capture method.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acid sequences present in a biologicalsample is important for identifying and classifying microorganisms,diagnosing infectious diseases, detecting and characterizing geneticabnormalities, identifying genetic changes associated with cancer,studying genetic susceptibility to disease, and measuring response tovarious types of treatment. A common technique for detecting andquantitating specific nucleic acid sequences is nucleic acidhybridization.

Various hybridization methods are available for the detection and studyof nucleic acids. In a traditional hybridization method, the nucleicacids to be identified are either in a solution or affixed to a solidcarrier. The nucleic acids are detected using labeled nucleic acidprobes which are capable of hybridizing to the nucleic acids. Recently,new hybridization methods have been developed to increase thesensitivity and specificity of detection. One example is the hybridcapture method described in U.S. application Ser. No. 07/792,585.Although these new hybridization methods offer significant improvementsover the traditional methods, they still lack the ability to fullydiscriminate between highly homologous nucleic acid sequences.

It is therefore an object of the present invention to provide ahybridization method which is not only rapid and sensitive, but is alsohighly specific and capable of discriminating highly homologous nucleicacid target sequences.

SUMMARY OF THE INVENTION

The present invention provides a novel nucleic acid detection method,referred to herein as target-specific hybrid capture (“TSHC”). TSHC is ahighly specific and sensitive method which is capable of discriminatingand detecting highly homologous nucleic acid target sequences.

In one embodiment, the method relates to detecting a target nucleic acidwherein the targeted nucleic acid, which is single-stranded or partiallysingle-stranded, is hybridized simultaneously, or sequentially, to acapture sequence probe and an unlabeled signal sequence probe. Theseprobes hybridize to non-overlapping regions of the target nucleic acidand not to each other so that double-stranded hybrids are formed. Thehybrids are captured onto a solid phase and detected. In a preferredembodiment, a DNA/RNA hybrid is formed between the target nucleic acidand the signal sequence probe. Using this method, detection may beaccomplished, for example, by binding a labeled antibody capable ofrecognizing a DNA/RNA hybrid to the double-stranded hybrid, therebydetecting the hybrid.

In another embodiment, the signal sequence probe used in the detectionmethod is a nucleic acid molecule which comprises a DNA/RNA duplex and asingle stranded nucleic acid sequence which is capable of hybridizing tothe single-stranded or partially single-stranded target nucleic acid.Detection may be accomplished, for example, by binding a labeledantibody capable of recognizing the DNA/RNA duplex portion of the signalsequence probe, thereby detecting the hybrid formed between the targetnucleic acid, the capture sequence probe and the signal sequence probe.

In yet another embodiment, the signal sequence probe used in thedetection method is a molecule which does not contain sequences that arecapable of hybridizing to the single-stranded or partiallysingle-stranded target nucleic acid. Bridge probes comprising sequencesthat are capable of hybridizing to the target nucleic acid as well assequences that are capable of hybridizing to the signal sequence probeare used. In this embodiment, the signal sequence probe comprises aDNA/RNA duplex portion and a single stranded DNA sequence portioncontaining sequences complementary to sequences within the bridge probe.The bridge probe, which hybridizes to both the target nucleic acid andthe signal sequence probe, therefore serves as an intermediate forconnecting the signal sequence probe to the target nucleic acid and thecapture sequence probe hybridized to the target nucleic acid.

In another embodiment of the TSHC method of the invention, blockerprobes comprising oligonucleotides complementary to the capture sequenceprobes are used in the method to eliminate excess capture sequenceprobe, thereby reducing the background signal in detection andincreasing specificity of the assay.

The present invention also relates to novel probes. These probes arenucleic acid sequences which can function in various hybridizationassays, including, for example, the TSHC assay.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram illustrating one embodiment of thetarget-specific hybrid capture method.

FIG. 2 is a schematic diagram illustrating one embodiment of thetarget-specific hybrid capture method.

FIG. 3 is a schematic diagram illustrating possible mechanisms of actionof an embodiment that employs fused capture sequence probes intarget-specific hybrid capture detection.

FIG. 4 shows the analytical sensitivity and specificity oftarget-specific hybrid capture detection of HSV-1.

FIG. 5 shows the analytical sensitivity and specificity oftarget-specific hybrid capture detection of HSV-2.

FIGS. 6A-6D show the various embodiments of the target-specific hybridcapture-plus method.

FIG. 7 shows the deletion probe embodiment of the target-specific hybridcapture method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting the presence ofnucleic acids in test samples. More specifically, the invention providesa highly specific and sensitive method which is capable ofdiscriminating and detecting highly homologous nucleic acid sequences.Preferred uses for this invention are well known to the skilled artisanand may be applied to the detection and discrimination of a variety ofmutations including, but not limited to insertions, deletions,inversions, repeated sequences, and multiple as well as singlenucleotide polymorphisms (SNPs). Additionally, this invention may alsobe group specific for the detection of nucleic acid targets that sharesimilar sequence elements.

Any source of nucleic acid, in purified or non-purified form, can beutilized as the test sample. For example, the test sample may be a foodor agricultural product, or a human or veterinary clinical specimen.Typically, the test sample is a biological fluid such as urine, blood,plasma, serum, sputum or the like. Alternatively the test sample may bea tissue specimen suspected of carrying a nucleic acid of interest. Thetarget nucleic acid in the test sample may be present initially as adiscrete molecule so that the sequence to be detected constitutes theentire nucleic acid, or may only be a component of a larger molecule. Itis not necessary that the nucleic acid sequence to be detected bepresent initially in a pure form. The test sample may contain a complexmixture of nucleic acids, of which the target nucleic acid maycorrespond to a gene of interest contained in total human genomic DNA orRNA or a portion of the nucleic acid sequence of a pathogenic organismwhich organism is a minor component of a clinical sample.

The target nucleic acid in a test sample can be DNA or RNA, such asmessenger RNA, from any source, including bacteria, yeast, viruses, andthe cells or tissues of higher organisms such as plants or animals.Methods for the extraction and/or purification of such nucleic acids arewell known in the art. Target nucleic acids may be double-stranded orsingle-stranded. In the present method, it is preferred that the targetnucleic acids are single-stranded or made single-stranded byconventional denaturation techniques prior to the hybridization steps ofthe method. In a preferred embodiment, base denaturation technique isused to denature the double-stranded target DNA.

The term “oligonucleotide” as the term is used herein refers to anucleic acid molecule comprised of two or more deoxyribonucleotides orribonucleotides. A desired oligonucleotide may be prepared by anysuitable method, such as purification from a naturally occurring nucleicacid, by molecular biological means, or by de novo synthesis. Examplesof oligonucleotides are nucleic acid probes described herein.

Nucleic acid probes are detectable nucleic acid sequences that hybridizeto complementary RNA or DNA sequences in a test sample. Detection of theprobe indicates the presence of a particular nucleic acid sequence inthe test sample. In one embodiment, the target-specific hybrid capturemethod employs two types of nucleic acid probes: capture sequence probe(CSP) and signal sequence probe (SSP). A capture sequence probecomprises a nucleic acid sequence which is capable of hybridizing tounique region(s) within a target nucleic acid and being captured onto asolid phase. A signal sequence probe comprises a nucleic acid sequencewhich is capable of hybridizing to regions within a target nucleic acidthat are adjacent to the unique regions recognized by the CSP. Thesequences of CSP and SSP are selected so that they would not hybridizeto the same region of a target nucleic acid or to each other.

In addition, the CSP and the SSP are selected to hybridize to regions ofthe target within 50, 000 bases of each other. The distance between thesequence to which the CSP hybridizes within the target nucleic acid andthe sequence to which the SSP hybridizes is preferably between 1 to50,000 bases, more preferably, the distance is less than 3,000 bases.Most preferably, the distance is less than 1,000 bases.

The CSP used in the detection method can be DNA, RNA, peptide nucleicacids (PNAs), locked nucleic acids (LNAs), or other nucleic acidanalogues. A “locked nucleic acid” as defined herein is a novel class ofoligonucleotide analogues which form duplexes with complementary DNA andRNA with high thermal stability and selectivity. The usualconformational freedom of the furanose ring in standard nucleosides isrestricted in LNAs due to the methylene linker connecting the 2′-Oposition to the 4′-C position. PNAs are oligonucleotides in which thesugar-phosphate backbone is replaced with a polyamide or “pseudopeptide”backbone. In a preferred embodiment, the CSP is DNA. The CSP has aminimum length of 6 bases, preferably between 15 to 100 bases long, andmore preferably between 20 to 40 bases long. The CSP is substantiallycomplementary to the sequence within a target nucleic acid to which ithybridizes. The sequence of a CSP is preferably at least 75%complementary to the target hybridization region, more preferably, 100%complementary to this sequence. It is also preferred that the CSPcontains less than or equal to 75% sequence identity, more preferablyless than 50% sequence identity, to non-desired sequences believed to bepresent in a test sample. The sequence within a target nucleic acid towhich a CSP binds is preferably 6 bases long, more preferably 20-40bases long. It may also be preferred that the sequences to which the CSPhybridizes are unique sequences or group-specific sequences.Group-specific sequences are multiple related sequences that formdiscrete groups.

In one embodiment, the CSP used in the detection method may contain oneor more modifications in the nucleic acid which allows specific captureof the probe onto a solid phase. For example, the CSP may be modified bytagging it with at least one ligand by methods well-known to thoseskilled in the art including, for example, nick-translation, chemical orphotochemical incorporation. In addition, the CSP may be tagged atmultiple positions with one or multiple types of labels. For example,the CSP may be tagged with biotin, which binds to streptavidin; ordigoxigenin, which binds to anti-digoxigenin; or 2,4-dinitrophenol(DNP), which binds to anti-DNP. Fluorogens can also be used to modifythe probes. Examples of fluorogens include fluorescein and derivatives,phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, Texas Red orother proprietary fluorogens. The fluorogens are generally attached bychemical modification and bind to a fluorogen-specific antibody, such asanti-fluorescein. It will be understood by those skilled in the art thatthe CSP can also be tagged by incorporation of a modified basecontaining any chemical group recognizable by specific antibodies. Othertags and methods of tagging nucleotide sequences for capture onto asolid phase coated with substrate are well known to those skilled in theart. A review of nucleic acid labels can be found in the article byLandegren, et al., “DNA Diagnostics-Molecular Techniques andAutomation”, Science, 242:229-237 (1988), which is incorporated hereinby reference. In one preferred embodiment, the CSP is tagged with biotinon both the 5′ and the 3′ ends of the nucleotide sequence. In anotherembodiment, the CSP is not modified but is captured on a solid matrix byvirtue of sequences contained in the CSP capable of hybridization to thematrix.

The SSP used in the detection method may be a DNA or RNA. In oneparticular embodiment of the invention, the SSP and target nucleic acidform a DNA/RNA hybrid. Therefore, in this embodiment, if the targetnucleic acid is a DNA, then the preferred SSP is an RNA. Similarly, ifthe target nucleic acid is RNA, then the preferred SSP is a DNA. The SSPis generally at least 15 bases long. However, the SSP may be up to orgreater than 1000 bases long. Longer SSPs are preferred. The SSP maycomprise a single nucleic acid fragment, or multiple smaller nucleicacid fragments each of which is preferably between 15 to 100 bases inlength.

In another embodiment, the SSP used in the detection method comprises aDNA/RNA duplex and a single stranded nucleic acid sequence capable ofhybridizing to the target nucleic acid (FIG. 6A). The SSP may beprepared by first cloning a single stranded DNA sequence complementaryto sequences within the target nucleic acid into a single-stranded DNAvector, then hybridizing RNA complementary to the DNA vector sequence togenerate a DNA/RNA duplex. For example, if M13 is used as the DNAvector, M13 RNA is hybridized to the M13 DNA sequence in the vector togenerate a DNA/RNA duplex. The resulting SSP contains a DNA/RNA duplexportion as well as a single stranded portion capable of hybridizing tosequences within the target nucleic acid. The single stranded DNA shouldbe at least 10 bases long, and may be up to or greater than 1000 baseslong. Alternatively, the DNA/RNA duplex portion of the SSP may be formedduring or after the reaction in which the single stranded portion of theSSP is hybridized to the target nucleic acid. The SSP can be linear,circular, or a combination of two or more forms. The DNA/RNA duplexportion of the SSP provides amplified signals for the detection ofcaptured hybrids using anti-DNA/RNA antibodies as described herein.

In yet another embodiment, the SSP used in the detection method is amolecule which does not contain sequences that are capable ofhybridizing to the target nucleic acid. In this embodiment, bridgeprobes comprising sequences capable of hybridizing to the target nucleicacid as well as sequences capable of hybridizing to the SSP are used.The bridge probes can be DNA, RNA, peptide nucleic acids (PNAs), lockednucleic acids (LNAs), or other nucleic acid analogues. In one embodiment(FIG. 6B), the SSP comprises a DNA/RNA duplex portion and a singlestranded portion containing sequences complementary to sequences withinthe bridge probe. The bridge probe, which is capable of hybridizing toboth the target nucleic acid and the SSP, therefore serves as anintermediate for connecting the SSP to the target nucleic acid and theCSP hybridized to the target nucleic acid. The SSP may be prepared asdescribed above. In another embodiment (FIG. 6C), the SSP used in thedetection method comprises multiple sets of repeat sequences as well asa single stranded RNA sequence capable of hybridizing to the bridgeprobe. A DNA oligonucleotide probe containing sequences complementary tothe repeat sequences may be used to hybridize to the SSP to generate theRNA/DNA duplex needed for signal amplification. In yet anotherembodiment (FIG. 6D), the bridge probe contains a poly(A) tail inaddition to sequences which are capable of hybridizing to the targetnucleic acid. The SSP used in this example comprises poly(dT) DNAsequences. The bridge probe therefore is capable of hybridizing to theSSP via its poly(A) tail. An RNA probe comprising poly(A) sequences maybe used to hybridize to the remaining poly(dT) DNA sequences within SSPto form an RNA/DNA duplex. The SSP comprising poly(dT) sequences and theRNA probe comprising poly(A) sequences are preferably 100 to 5,000 baseslong.

The SSP used in the detection method of the invention can be unmodified,or modified as with the CSP using methods described above and/or knownin the art. In a preferred embodiment, the SSP is a covalentlyunmodified probe.

It is understood that multiple CSPs and/or SSPs can be employed in thedetection method of the invention.

In another embodiment, an oligonucleotide probe comprising complementarysequences of two or more distinct regions of the target nucleic acid arefused together and used as the capture sequence probe in the method ofthe invention. Alternatively a single probe can be designed and producedwhich contains sequences complementary to single or multiple targetnucleic acids. This type of probe is also referred to herein as a“fused” CSP. As shown in Example 5, the fused capture sequence probeworks as effectively as the combination of two unfused CSPs when used atthe same concentration.

In a further embodiment of this invention, “deletion nucleic acidprobes” may be used in TSHC. In order to minimize the number oftranscription templates needed to be constructed, deletion nucleic acidprobes, for example RNA, are designed such that 1) the length of probeused is maximized; and 2) probes are prevented from overlapping with theregion targeted by the CSP. These deletion probes contain internaldeletions in the nucleic acid template used to generate the probes. Inaddition, these deletion probes hybridize to nucleic acid targetscreating “bubbles” of unhybridized nucleic acid that is accessible forCSP hybridization. This method also provides a very convenient means formaking probes since the nucleic acid for the entire target may be clonedinto a transcription vector and then sequences may be removed once theyhave been identified as useful regions for CSP hybridization. Inaddition, this method permits the use of nearly full length whole genomeprobes that do not overlap (i.e. do not hybridize to the same region)with the CSPs. Any commercially available mutagenesis kit can be used todesign targeted deletions within a transcription template. Typically,the deletions of the nucleic acid template used for SSP synthesis areperformed directly with the template cloned in the transcription vector.Deletions in the template are made such that the sequences overlappingthe region hybridized by the CSP are removed. The deletions may be assmall as the CSP region itself, but generally and more preferably,approximately 100 to 300 nucleotides on the 5′ and 3′ ends of the regionhybridized by the CSP are deleted. (See FIG. 7).

The nucleic acid probes of the invention may be produced by any suitablemethod known in the art, including for example, by chemical synthesis,isolation from a naturally-occurring source, recombinant production andasymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods andapplications. San Diego, Calif., Academic Press, 76-83). It may bepreferred to chemically synthesize the probes in one or more segmentsand subsequently link the segments. Several chemical synthesis methodsare described by Narang et al. (1979 Meth. Enzymol. 68:90), Brown et al.(1979 Meth. Enzymol. 68:109) and Caruthers et al. (1985 Meth. Enzymol.154:287), which are incorporated herein by reference. Alternatively,cloning methods may provide a convenient nucleic acid fragment which canbe isolated for use as a promoter primer. A double-stranded DNA probe isfirst rendered single-stranded using, for example, conventionaldenaturation methods prior to hybridization to the target nucleic acids.

Hybridization is conducted under standard hybridization conditions wellknown to those skilled in the art. Reaction conditions for hybridizationof a probe to a nucleic acid sequence vary from probe to probe,depending on factors such as probe length, the number of G and Cnucleotides in the sequence, and the composition of the buffer utilizedin the hybridization reaction. Moderately stringent hybridizationconditions are generally understood by those skilled in the art asconditions approximately 25° C. below the melting temperature of aperfectly base-paired double stranded DNA. Higher specificity isgenerally achieved by employing incubation conditions having highertemperatures, in other words more stringent conditions. Chapter 11 ofthe well-known laboratory manual of Sambrook at al., MOLECULAR CLONING:A L ABORATORY MANUAL, second edition, Cold Spring Harbor LaboratoryPress, New York (1990) (which is incorporated by reference herein),describes hybridization conditions for oligonucleotide probes in greatdetail, including a description of the factors involved and the level ofstringency necessary to guarantee hybridization with specificity.Hybridization is typically performed in a buffered aqueous solution, forwhich conditions such as temperature, salt concentration, and pH areselected to provide sufficient stringency such that the probes hybridizespecifically to their respective target nucleic acid sequences but notany other sequence.

Generally, the efficiency of hybridization between probe and targetimprove under conditions where the amount of probe added is in molarexcess to the template, preferably a 2 to 10⁶ molar excess, morepreferably 10³ to 10⁶ molar excess. The concentration of each CSPprovided for efficient capture is at least 25 fmoles/ml (25 pM) in thefinal hybridization solution, preferably between 25 fmoles to 10⁴fmoles/ml (10 nM). The concentration of each SSP is at least 15 ng/ml inthe final hybridization solution, preferably 150 ng/ml. Table A showsthe conversion of SSP concentrations expressed in ng/ml to molar basis.

TABLE A CONVERSION OF SSP CONCENTRATION FROM ng/ml TO fmoles/ml SSPConcentration SSP Concentration in fmoles/ml (pM) in ng/ml SSP is a 3 kbRNA SSP is a 5 kb RNA  15 ng/ml 15.1 9 150 ng/ml 151 90 600 ng/ml 606364

Hybridization of the CSP and the SSP to the target nucleic acid may beperformed simultaneously or sequentially and in either order. In oneembodiment, hybridization of the CSP and hybridization of the SSP to thetarget nucleic acid are performed simultaneously. The hybrid formed isthen captured onto a solid phase coated with a substrate to which ligandattached to the CSP binds with specificity. In another embodiment,hybridization of the SSP to the target nucleic acid is performed afterthe hybridization of the CSP to the target nucleic acid. In this case,the CSP may be immobilized on a solid phase before or afterhybridization. In this embodiment, both the CSP and the target may bebound to the solid phase during the SSP hybridization reaction. Mostpreferably, the CSP and SSP are hybridized to the target nucleic acid,forming a hybridized complex, wherein said complex is then captured ontoa solid phase coated with a substrate to which ligand attached to theCSP binds with specificity.

In order to identify and detect specific polynucleotide sequences withadded specificity and sensitivity, assays can be designed such thatconditions are optimal for increasing signal detection and reducingbackground interference. Preferred methods for achieving addedstringency include the TSHC heated capture step and/or through the useof blocker probes. Since capture efficiency of the hybridized complexcomprising CSP, SSP, and target nucleic acid is influenced by severalassay conditions, a heated capture may be useful for reducing falsereactivity and detecting mutations of at least one nucleotide.Preferably, the heated capture method is employed for the detection ofsingle nucleotide polymorphisms. Briefly, the heated capture method forcapturing or binding the hybridized complex to a solid phase utilizes anelevated range of temperatures. In order to immobilize CSP hybridizedtargets following hybridization, the hybridization solution is placedinto wells of a 96-well plate, for example, and the plate is shaken for15 minutes to 2 hours at temperatures ranging from 20° C. to 90° C.shaking at 1100 rpms. Optionally, hybridization at room temperature for1 hour shaking at 1100 rpms may be preferred. Capture temperatures aboveroom temperature may be preferred for an added level of stringency ashybridization (and “promiscuous hybridization”) does occur during theplate capture step. Another means for attaining a higher degree ofspecificity and sensitivity is through the use of blocker probes.

One embodiment of this invention provides a heated capture method usingelevated temperatures for capturing a hybridized SSP and target nucleicacid complex to a CSP immobilized to a solid phase, eithersimultaneously or sequentially, where the elevated temperature preventsnon-specific hybridization of the CSP from occurring during the platecapture step. The elevated temperature also affects SSP hybridizationspecificity. The CSP used in TSHC may be a nucleic acid or modifiednucleic acid, preferably DNA, which contains a modification that permitscapture onto a solid phase. One example of such a modification is abiotin label and more preferably multiple biotin labels. The CSPcontains a minimum of 6 base pairs, preferably 16 to 50 bases with apreferred melting temperature (Tm) above 65° C. Preferred CSPs maycomprise sequences complementary to unique sequences within the targetmolecule of nucleic acids present in the sample, although this is notnecessary for targeting multiple nucleic acid species. For example, if agene family is the target, the CSP may preferably comprise a sequenceelement common to one or more members of the gene family. For mostapplications, the CSP preferably contains at most 75% sequence identityand more preferably less than 50%, to non-desired targets suspected ofbeing present in the sample. The assay can utilize CSPs that differ inonly a single nucleotide and selectively detect targets that differ inonly a single nucleotide. This degree of discrimination can befacilitated by using the heated plate capture step. When CSPhybridization is performed in solution, the samples are subsequentlyreacted with a solid phase for capture. For example, if a biotin-labeledCSP is used, avidin or some other biotin binding protein may be used tocoat the solid phone for capture. Another embodiment of this inventionencompasses the simultaneous hybridization and capture, whereinhybridization is performed directly on the capture solid phase, forexample on a capture plate.

In yet another embodiment of this invention, the TSHC method can be usedto distinguish and detect nucleic acid targets with SNPs. This HybridCapture-Single Nucleotide Polymorphism (HC-SNP) detection method candetect SNPs with high sensitivity and specificity. An exampleillustrating the extended capability of TSHC for distinguishing anddetecting nucleic acid targets with SNPs is described herein, where inlabeled capture oligonucleotides (CSP) are used, in addition to signalsequence probes (SSP), and a target nucleic acid molecule. The CSPs mayhybridize and capture nucleic acid targets to a solid phase or surface(for example, a 96-well plate). Labeling methods are well known in theart and may also be employed to facilitate target nucleic acidimmobilization.

In one example, a target nucleic acid capture is achieved through thehigh affinity interaction between a biotin on the CSP and a streptavidinon the solid surface. Simultaneously, an RNA signal sequence probe (SSP)complementary to a DNA target and not overlapping with the captureregion is hybridized to the DNA target. The RNA/DNA hybrids arerecognized by antibody directed against RNA/DNA hybrids labeled withalkaline phosphatase. In this example, a chemiluminescent phosphorylatedsubstrate is then added and subsequently, the activated substrate may bedetected and measured by a luminometer. The signal to noise ratios aredetermined using a known negative control. Further, the concentration ofthe target can be determined by using known concentrations of targetmolecules as calibrators. The specificity of binding and capturing thehybrid to a solid phase is modulated, regulated, or adjusted bytemperatures of greater than room temperature, by the addition ofblocker probes, or by temperatures of greater than room temperature andthe addition of blocker probes. For additional stringency, blockerprobes may be used either with or without the heated capture method.Alternatively, the capture step may be performed at room temperature andmay optionally utilize blocker probes.

Another embodiment of this invention further provides a blockeroligonucleotide method where in many cases, obviates the need for aheated capture step. This may be achieved by hybridizing blockeroligonucleotides to capture oligonucleotides at room temperature,thereby preventing further hybridization of the CSP to undesired targetsduring the capture step. Capture probes may preferably require thepresence of blocker probes, which are complementary to the captureprobe. The length of the blocker probes can vary from blockerscomplementary to the full length CSP to very short blockerscomplementary to only a small portion of the CSP. For example, blockerprobes can be 4-10 base pairs shorter than the length of the CSP. Thepresence of the blocker probes reduces background and enables a higherdegree of sensitivity. The heated capture step and blocker probes may beused either separately or together, wherein the specificity of bindingand capturing the hybrid to a solid phase is modulated, regulated, oradjusted by temperatures of greater than room temperature and theaddition of blocker probes.

It will be understood by those skilled in the art that a solid phase ormatrix includes, for example, polystyrene, polyethylene, polypropylene,polycarbonate or any solid plastic material in the shape of plates,slides, dishes, beads, particles, microparticles, cups, strands, chipsand strips. A solid phase also includes glass beads, glass test tubesand any other appropriate glass product. A functionalized solid phasesuch as plastic or glass that has been modified so that the surfacecontains carboxyl, amino, hydrazide, aldehyde groups, nucleic acid ornucleotide derivatives can also be used. Any solid phase such as plasticor glass microparticles, beads, strips, test tubes, slides, strands,chips or micsotiter plates can be used.

In one preferred embodiment, the CSP is labeled with biotin, andstreptavidin-coated or avidin-coated solid phase is employed to capturethe hybrid. More preferably, streptavidin-coated microliter plates areused. These plates may be coated passively or covalently.

The captured hybrid may be detected by conventional means well-known inthe art, such as with a labeled polyclonal or monoclonal antibodyspecific for the hybrid, an antibody specific for one or more ligandsattached to the SSP, a labeled antibody, or a detectable modification onthe SSP itself.

One preferred method of detection detects the captured hybrid by usingan antibody capable of binding to the RNA/DNA hybrid (referred to hereinas the “RNA/DNA antibody”. In this embodiment, the anti-RNA/DNA antibodyis preferably labeled with an enzyme, a fluorescent molecule or abiotin-avidin conjugate and is non-radioactive. The label can bedetected directly or indirectly by conventional means known in the artsuch as a colorimeter, a luminometer, or a fluorescence detector. Onepreferred label is, for example, alkaline phosphatase. Other labelsknown to one skilled in the art can also be employed as a means ofdetecting the bound double-stranded hybrid.

Detection of captured hybrid is preferably achieved by binding theconjugated antibody to the hybrid during an incubation step. Surfacesare then washed to remove any excess conjugate. These techniques areknown in the art. For example, manual washes may be performed usingeither an Eppendorf™ Repeat Pipettor with a 50 ml Combitip™ (EppendorfHamburg, Germany), a Corning repeat syringe (Corning, Corning, N.Y.), asimple pump regulated by a variostat, or by gravity flow from areservoir with attached tubing. Commercially available tube washingsystems available from Source Scientific Systems (Garden Grove, Calif.)can also be used.

Bound conjugate is subsequently detected by a method conventionally usedin the art, for example, colorimetry or chemiluminescence as describedat Coutlee, et al., J. Clin. Microbiol. 27:1002-1007 (1989). Preferably,bound alkaline phosphatase conjugate is detected by chemiluminescence byadding a substrate which can be activated by alkaline phosphatase.Chemiluminescent substrates that are activated by alkaline phosphataseare well known in the art.

In another embodiment, the target specific hybrid capture Method of theinvention employs blocker probes in addition to the CSP and SSP. Ablocker probe comprises sequences that are complementary to thesequences of the CSP. The sequence of a blocker probe is preferably atleast 75% complementary to the sequence of the CSP, more preferably,100% complementary to the CSP. The addition of the blocker probes to thehybridization reaction mixture prevents non-hybridized CSP fromhybridizing to cross-reactive nucleic acid sequences present in thetarget and therefore increases the specificity of the detection.

The blocker probe is generally at least 5 bases long, preferably 12bases long. The concentration of the blocker probe in the hybridizationreaction is preferably in excess to that of the CSP and SSP. Preferably,the blocker probe is present in a 2-fold molar excess, although, it maybe present in an up to 10,000-fold molar excess. The blacker probes canbe DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acidanalogues.

In one embodiment, blocker probes complementary to the full-length ornear full-length of the CSP are used. Following the reaction in whichthe hybrid between CSP, SSP and the target nucleic acid is formed, oneor more blocker probes may be added to the reaction and thehybridization is continued for a desired time. The hybridizationproducts are then detected as described above.

In another embodiment, blocker probes complementary to only a portion ofthe CSP and shorter than the CSP are used. These blocker probes have alower melting temperature than that of the CSP. Preferably, the meltingtemperature of the blocker probe is 10 degrees lower than that of theCSP. In this case, the blocker probe is preferably added to the targetnucleic acids simultaneously with the CSP and the SSP. Since the blockerprobe has a lower melting temperature than the CSP, the initialtemperature for hybridization is chosen such that the blocker probe doesnot interfere with the hybridization of the CSP to its target sequences.However, when the temperature of the hybridization mixtures is adjustedbelow the temperature used for target hybridization, the blocker probehybridizes to the CSP and effectively blocks the CSP from hybridizing tocross-reactive nucleic acid sequences. For example, when thehybridization products are incubated at room temperature on astreptavidin-coated microtiter plate during hybrid capture, the blockerprobes may be added.

The following examples illustrate use of the present amplificationmethod and detection assay and kit. These examples are offered by way ofillustration, and are not intended to limit the scope of the inventionin any manner. All references described herein are expresslyincorporated in tow by reference.

EXAMPLE 1 Target-Specific Hybrid Capture (TSHC) Assay Protocol

Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) viralparticles of known concentration (Advanced Biotechnologies, Inc.,Columbia, Md.) or clinical samples were diluted using either NegativeControl Media (Digene Corp., Gaithersburg, Md.) or Negative CervicalSpecimens (Digene). Various dilutions were made and aliquoted intoindividual microfuge tubes. A half volume of the Denaturation Reagent5100-0431 (Digene) was added. Test samples were incubated at 65° C. for45 minutes for denaturation of nucleic acids in the samples.

Following denaturation, a hybridization solution containing signalsequence probes (SSPs) (600 ng/ml each) and capture sequence probes(CSPs) (2.5 pmoles/ml each) was added to the sample, and incubated at74° C. for 1 hour. Blocker probes in a solution containing one volume of4× Probe Diluent (Digene), one volume of Denaturation Reagent, and twovolumes of the Negative Control Media were then added to thehybridization mixture and incubated at 74° C. for 15 minutes.

In a second series of experiments, following denaturation of nucleicacids, a hybridization mixture containing SSPs (600 ng/ml each), CSPs(2.5 pmoles/ml each), and blocker probes (250 pmoles/ml each) was addedto the samples and incubated for one hour at 74° C.

Tubes containing reaction mixtures were cooled at room temperature for 5minutes, and aliquots were taken from each tube and transferred toindividual wells of a 96-well streptavidin capture plate (Digene). Theplates were shaken at 1100 rpms for 1 hour at room temperature. Thesupernatants were then decanted and the plates were washed twice withHybrid Capture 2 wash buffer (Digene) and inverted briefly to removeresidual wash buffer. The alkaline-phosphatase anti-RNA/DNA antibodydetection reagent-1 (DR-1; Digene) was then added to each well andincubated for 30 minutes at room temperature (about 20° C. to 25° C.).The wells were then subjected to multiple wash steps which include: 1)three washes with Sharp wash buffer (Digene) at room temperature; 2)incubation of the plate with the Sharp wash buffer for 10 minutes at 60°C. on a heat block; 3) two washes with the Sharp wash buffer at roomtemperature; and 4) one wash with the SNM wash buffer (Digene) at roomtemperature. Following removal of the residual liquid, luminescentsubstrate 5100-0350 (Digene) was added to each well and incubated for 15minutes at room temperature. The individual wells were then read on aplate luminometer to obtain the relative light unit (RLU) signal.

Solutions containing Negative Control Media or known HSV NegativeCervical Specimens were used as negative controls for the test samples.The signal to noise ratio (S/N) was calculated as the ratio of theaverage RLU obtained from a test sample to the average RLU of thenegative control. The signal to noise ratio was used as the basis fordetermining capture efficiency and the detection of target nucleicacids. A S/N value of 2 or greater was arbitrarily assigned as apositive signal while a S/N value less than 2 was considered negative.The coefficient of variation (CV) which is a determination of thevariability of the experiment within one sample set was calculated bytaking the standard deviation of the replicates, dividing them by theaverage and multiplying that value by 100 to give a percent value.

The capture sequence probes and the blocker probes used in experimentsdescribed in Examples 2-13 were synthesized using the method describedby Cook et al. (1988 Nucl. Acid. Res., 16: 4077-95). Unless otherwisenoted, the capture sequence probes used in the experiments describedherein were labeled with biotins at their 5′ and 3′ ends.

The signal sequence probes used in experiments described in Examples2-13 are RNA probes, but this invention is not limited to SSPscomprising RNA. These probes were prepared using the method described byYisraeli et al. (1989, Methods in Enzymol., 180: 42-50).

EXAMPLE 2

The following tables describe the various probes used in experimentsdescribed in Examples 3-13.

TABLE 1 HSV-1 CLONES FROM WHICH HSV-1 PROBES ARE DERIVED Insert SequenceClone Size Location Name Host Vector Cloning Site(s) (bp) within HSV-1RH3 Dgx3 Hind III, Eco RI 5720 39850-45570 R10 Blue Script SK+ Eco RI4072 64134-68206 RH5B Blue Script SK+ Eco RV, Eco RI 4987 105108-110095H19 Blue Script SK+ Hind III 4890 133467-138349

TABLE 2 CLONES FROM WHICH HSV-2 PROBES ARE DERIVED Insert Sequence CloneSize Location Name Host Vector Cloning Site(s) (bp) in HSV-2 E4A BlueScript SK+ Bam HI 3683 23230-26914 E4B Blue Script SK+ Bam HI Eco RI5600 26914-32267 I8 Blue Script SK+ Hind III 2844 41624-44474 EI8 Dgx3Hind III, Eco RI 3715 44474-48189 4L Blue Script KS+ Bam HI, Eco RI 431386199-90512

TABLE 3 CAPTURE SEQUENCE PROBES FOR HSV-1 Location Size within ProbeSequence (bP) HSV-1 TS-1 (TTATTATTA)CGTTCATGTCGGCAAACAGCT 24 105040-CGT(TTATTATTA) [SEQ ID NO: 1] 105063 TS-2(TTATTATTA)CGTCCTGGATGGCGATACGGC 21 110316- (TTATTATTA) [SEQ ID NO: 2]110336 VH-3 CGTCCTGGATGGCGATACGGC [SEQ ID NO: 3] 21 110316- 110336 NC-1CGTTCATGTCGGCAAACAGCTCGT [SEQ ID NO: 4] 24 105040- 105063 VH-4CGTTCATGTCGGCAAACAGCTCGT- 45 105040- (fusion ofCGTCCTGGATGGCGATACGGC [SEQ ID NO: 5] 105063; VH3, NC-1) 110316- 110336HZ-1 GATGGGGTTATTTTTCCTAAGATGGGGC 34 133061- GGGTCC [SEQ ID NO: 6]133094 VH-2 TACCCCGATCATCAGTTATCCTTAAGGT [SEQ ID 28 138367- NO: 7]138394 FD-1 AAACCGTTCCATGACCGGA [SEQ ID NO: 8] 19 39281-39299 RA-2ATCGCGTGTTCCAGAGACAGGC [SEQ ID NO: 9] 22 39156-39177 NC-2CAACGCCCAAAATAATA [SEQ ID NO: 10] 17 46337-46353 FD-2GTCCCCGAaCCGATCTAGCG (note small cap a is 20 45483-45502mutated base) [SEQ ID NO: 11] RA-4CGAACCATAAACCATTCCCCAT [SEQ ID NO: 12] 22 46361-46382 ON-3CACGCCCGTGGTTCTGGAATTCGAC [SEQ ID 25 64105-64129 NO: 13] HZ-2(TTTATTA)GATGGGGTTATTTTTCCTAAGATGGGG 34 133061- CGGGTCC [SEQ ID NO: 14]133094 ZD-1 GGTTATTTTTCCTAAG [SEQ ID NO: 15] 16 133064- 133079 ZD-2(ATTATT)GGTTATTTTTCCTAAG(ATTATT) [SEQ ID 16 133064- NO: 16] 133079 F6RACGACGCCCTTGACTCCGATTCGTCATCGGATGA 40 87111-87150 CTCCCT [SEQ ID NO: 17]BRH19 ATGCGCCAGTGTATCAATCAGCTGTTTCGGGT 32 133223- [SEQ ID NO: 18] 133254F15R CAAAACGTCCTGGAGACGGGTGAGTGTCGGCGAG 38 141311- GACG [SEQ ID NO: 19]141348 VH-1 GTCCCCGACCCGATCTAGCG [SEQ ID NO: 20] 20 45483-45502 ON-4GCAGACTGCGCCAGGAACGAGTA [SEQ ID NO: 21] 23 68404-68426 PZ-1GTGCCCACGCCCGTGGTTCTGGAATTCGACAGCG 35 64105-64139 A [SEQ ID NO: 22] PZ-2GCAGACTGCGCCAGGAACGAGTAGTTGGAGTACT 35 68404-68438 G [SEQ ID NO: 23] FG-2AAGAGGTCCATTGGGTGGGGTTGATACGGGAAAG 36 105069- AC [SEQ ID NO: 24] 105104FG-3 CGTAATGCGGCGGTGCAGACTCCCCTG-[SEQ ID 27 110620- NO: 25] 110646 FG-4CCAACTACCCCGATCATCAGTTATCCTT 39 138362- AAGGTCTCTTG [SEQ ID NO: 26]138400 Hsv1-LF15R (AAAAAAAAA)CAAAACGTCCTGGAGACGGGTGA 38 141311-141348(SH-3) GTGTCGGCGAGGACG [SEQ ID NO: 27] Hsv1-F15-28CAAAACGTCCTGGAGACGGGTGAGTGTCGGCGAG 38 141311-141348 (GZ-1)GACG [SEQ ID NO: 28] Hsv1-F15-3B CAAAACGTCC-bio-U-GGAGACGGGTGAG 38141311-141348 (GZ-2) TG-bio-U-CGGCGAGGACG [SEQ ID NO: 29] *Sequences inparentheses are “tail” sequences not directed at HSV.

TABLE 4 BLOCKER PROBES FOR HSV-1 Size Capture Probe to Probe Sequence(bp) which it hybridizes EA-1 AGGAAAAATAACCCCATC [SEQ ID NO: 30] 18 HZ-1EA-2 GACCCGCCCCATCTT [SEQ ID NO: 31] 15 HZ-1 ZD-3GGACCCGCCCCATCTTAGGAAAAATAAC 34 HZ-1 CCCATC [SEQ ID NO: 32] NG-7AAAAATAACCCCA [SEQ ID NO: 33] 13 HZ-1 NG-8 CGCCCCATCTT [SEQ ID NO: 34]11 HZ-1 NG-4 CCATCTTAGGAAAAA [SEQ ID NO: 35] 15 HZ-1 GP-1ATAACTGATGATCGG [SEQ ID NO: 36] 15 VH-Z EA-3CCACCCAATGGACCTC [SEQ ID NO: 37] 16 FG-2 EA-4GTCTTTCCCGTATCAACC [SEQ ID NO: 38] 18 FG-2 EB-7CGCCGCATTACG [SEQ ID NO: 39] 12 FG-3 EB-8 AGGGGAGTCTGC [SEQ ID NO: 40]12 FG-3 GP-3 CTGTTTGCCGACA [SEQ ID NO: 41] 13 VH-4 GP-4TATCGCCATCCAG [SEQ ID NO: 42] 13 VH-4 EB-9ATGATCGGGGTAGT [SEQ ID NO: 43] 14 FG-4  EB-10AGAGACCTTAAGGATA [SEQ ID NO: 44] 16 FG-4 NG-1ATTCCAGAACCACGG [SEQ ID NO: 45] 15 ON-3 NG-2TTCCAGAACCACG [SEQ ID NO: 46] 13 ON-3 NG-3 TCCAGAACCAC [SEQ ID NO: 47]11 ON-4 GP-5 GTTCCTGGCGCAG [SEQ ID NO: 48] 13 ON-4 GP-6TTCCTGGCGCAG [SEQ ID NO: 49] 12 ON-4

TABLE 5 CAPTURE SEQUENCE PROBES FOR HSV-2 Size Location within ProbeSequence (bp) HSV-2 NF-1 GCCCGCGCCGCCAGCACTACTTTC 24 41610-41587[SEQ ID NO: 50] FG-1 AAACGTTGGGAGGTGTGTGCGTCATCCTG 35 48200-48234GAGCTA [SEQ ID NO: 51] LE-3 GACCAAAACCGAGTGAGGTTCTGTGT 26 48732-48757[SEQ ID NO: 52] NF-2 AAACGTTGGGAGGTGTGTGCGTCA  24 48200-48223[SEQ ID NO: 53] RA-3 TGCTCGTCACGAAGTCACTCATG 23 22756-22734[SEQ ID NO: 54] ON-2 CATTACTGCCCGCACCGGACC 21 23862-23842[SEQ ID NO: 55] LE-1 GCCGTGGTGTTCCTGAACACCAGG 24 27666-27643[SEQ ID NO: 56] LE-4 AGTCAGGGTTGCCCGACTTCGTCAC 25 22891-22867[SEQ ID NO: 57] NF-3 CAGGCGTCCTCGGTCTCGGGCGGGGC 26 32847-32822[SEQ ID NO: 58] NF-4 CCCACGTCACCGGGGGCCCC 20 26743-26724 [SEQ ID NO: 59]LE-2 GCCGGTCGCGTGCGACGCCCAAGGC 25 33130-33106 [SEQ ID NO: 60] SG-3CCGACGCGTGGGTATCTAGGGGGTCG 26 90559-90534 [SEQ ID NO: 61] SG-4CGGGACGGCGAGCGGAAAGTCAACGT 26 86194-86169 [SEQ ID NO: 62]

TABLE 6 BLOCKER PROBES FOR HSV-2 Size Capture Probe to Probe NameSequence (bp) which it hybridizes HX-4 GGCGCGGGC [SEQ ID NO: 63]  9 NF-1HX-5 GAAAGTAGTGCTGGC [SEQ ID NO: 64] 15 NF-1 GP-7TGCTGGCGGCG [SEQ ID NO: 65] 11 NF-1 AZ-3 ACACCTCCCAACG [SEQ ID NO: 66]13 FG-1 AZ-4 CTCCAGGATGACG [SEQ ID NO: 67] 13 FG-1 GR-1TCGGTTTTGGTC [SEQ ID NO: 68] 12 LE-3 GR-2 ACACAGAACCTCA [SEQ ID NO: 69]13 LE-3 GP-8 CACACACCTCCCA [SEQ ID NO: 70] 13 NF-2 BR-10CGACCCCCTAGATA [SEQ ID NO: 71] 14 SG-3 BR-11 CCACGCGTCGG [SEQ ID NO: 72]11 SG-3 HX-6 ACGTTGACTTTCCGC [SEQ ID NO: 73] 15 SG-4 BR-15CGCCGTCCCG [SEQ ID NO: 74] 10 SG-4

TABLE 7 CAPTURE SEQUENCE PROBES FOR HPV Size HPV Type and Probe Sequence(bp) Sequence Location ZL-1 GTACAGATGGTACCGGGGTTGTAGAAGTATCTG 33 HPV16[SEQ ID NO: 75] 5360-5392 ZL-4 CTGCAACAAGACATACATCGACCGGTCCACC 31 HPV16[SEQ ID NO: 76] 495-525 DP-1 GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG 31 HPV16[SEQ ID NO: 77] 5285-5315 DP-4 CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG 33HPV16 [SEQ ID NO: 78] 128-160 SH-1 GAGGTCTTCTCCAACATGCTATGCAACGTCCTG 33HPV31 [SEQ ID NO: 79] 505-537 SH-4 GTGTAGGTGCATGCTCTATAGGTACATCAGGCC 33HPV31 [SEQ ID NO: 80] 5387-5419 VS-1 CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG33 HPV31 [SEQ ID NO: 81] 132-164 VS-4 GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC33 HPV31 [SEQ ID NO: 82] 5175-5207 AH-1 GAACGCGATGGTACAGGCACTGCAGGGTCC30 HPV18 [SEQ ID NO: 83] 5308-5337 AH-2 GAACGCGATGGTACAGGCACTGCA [SEQ ID24 HPV18 NO: 84] 5314-5337 AL-1 ACGCCCACCCAATGGAATGTACCC [SEQ ID 24HPV18 NO: 85] 4451-4474 PA-4 TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC 32 HPV18[SEQ ID NO: 86] 535-566 18-1AB (TTATTATTA)CTACATACATTGCCGCCATGTTCG 36KPV18 CCA [SEQ ID NO: 87] 1369-1395 18-2AB(TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGT 46 HPV18CTATAGCCTCCGT [SEQ ID NO: 88] 1406-1442 18-3AB(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAA 38 HPV18 GTTTGC [SEQ ID NO: 89]7524-7552 18-4AB (TTATTATTA)CACGGTGCTGGAATACGGTGAGG 37 HPV18GGGTG [SEQ ID NO: 90] 3485-3512 18-5AB(TTATTATTA)ACGCCCACCCAATGGAATGTACCC 33 HPV18 [SEQ ID NO: 91] 4451-447418-6AB (TTATTATTA)ATAGTATTGTGGTGTGTTTCTCAC 35 HPV18 AT [SEQ ID NO: 92]81-106 18-7AB (TTATTATTA)GTTGGAGTCGTTCCTGTCGTG 30 HPV18 [SEQ ID NO: 93]538-558 18-8AB (TTATTATTA)CGGAATTTCATTTTGGGGCTCT 31 HPV18[SEQ ID NO: 94] 634-655 PE-1 GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT 33 HPV18[SEQ ID NO: 95] 811-843 PZ-2 GCGCCATCCTGTAATGCACTTTTCCACAAAGC 32 HPV45[SEQ ID NO: 96] 77-108 PZ-5 TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG 31 HPV45[SEQ ID NO: 97] 5295-5325 CS-1 GGTCACAACATGTATTACACTGCCCTCGGTAC 32 HPV45[SEQ ID NO: 98] 500-531 CS-4 CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC 31 HPV45[SEQ ID NO: 99] 533-563 PF-1 CTGCATTGTCACTACTATCCCCACCACTACTTTG 34 HPV45[SEQ ID NO: 100] 1406-1439 PF-4 CCACAAGGCACATTCATACATACACGCACGCA 32HPV45 [SEQ ID NO: 101] 7243-7274 PA-1 GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA33 HPV45 [SEQ ID NO: 102] 811-843 45-5AB(TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGAGGC 36 HPV45 [SEQ ID NO: 103]3444-3470 45-6AB (TTATTATTA)AGACCTGCCCCCTAAGGGTACATAGCC 36 HPV45[SEQ ID NO: 104] 4443-4469 45-8AB (TTATTATTA)CAGCATTGCAGCCTTTTTGTTACT 49HPV45 TGCTTGTAATAGCTCC [SEQ ID NO: 105] 1477-1516 45-9AB(TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA 34 HPV45 [SEQ ID NO: 106]  79-10345-10AB (TTATTATTA)GCCTGGTCACAACATGTATTAC 31 HPV45 [SEQ ID NO: 107)514-535 45-11AB (TTATTATTA)CAGGATCTAATTCATTCTGAGGTT 33 HPV45[SEQ ID NO: 108] 633-656 ON-1 TGCGGTTTTGGGGGTCGACGTGGAGGC [SEQ ID 27HPV45 NO: 109] 3444-3470 *Sequences in parentheses are “tail” sequencesnot directed at HSV.

TABLE 8 BLOCKER PROBES FOR HPV Capture Probe to Size which it ProbeSequence (bp) hybridizes PV-FD-1 GCCTCCACGTCGAC [SEQ ID NO: 110] 14ON-1/45-5AB PV-FD-2 CCCCAAAACCG [SEQ ID NO: 111] 11 ON-1/45-5AB PV-FD-3GGTACATTCCATTGGG [SEQ ID NO: 112] 16 18-5AB/AL-1 PV-FD-4TGGGCGTTAATAATAA [SEQ ID NO: 113] 16 18-5AB AH-3ACCATCGCGTTC [SEQ ID NO: 114] 12 AH-2 AH-4GGACCCTGCAGTGC [SEQ ID NO: 115] 14 AH-1 AH-5CTGTACCATCGCGTT 3′[SEQ ID NO: 116] 15 AH-1 AH-6TGCAGTGCCTGT [SEQ ID NO: 117] 12 AH-2 PZ-1CCACCTCCTGCGT [SEQ ID NO: 118] 13 PZ-5 PZ-3ATTACAGGATGGCGC [SEQ ID NO: 119] 15 PZ-2 PZ-4GCTTTGTGGAAAAGTG [SEQ ID NO: 120] 16 PZ-2 PZ-6CCACTACACCTAGCACTA [SEQ ID NO: 121] 18 PZ-5 ZL-2CAGATACTTCTACAACC [SEQ ID NO: 122] 17 ZL-1 ZL-3CCGGTACCATCTGTAC [SEQ ID NO: 123] 16 ZL-1 ZL-5GGTGGACCGGTCG [SEQ ID NO: 124] 13 ZL-4 ZL-6ATGTATGTCTTGTTGCAG [SEQ ID NO: 125] 18 ZL-4 DP-2CTACCACTTCACATGC [SEQ ID NO: 126] 16 DP-1 DP-3AGCCTCACCTACTTC [SEQ ID NO: 127] 15 DP-1 DP-5CCCAGAAAGTTACCAC [SEQ ID NO: 128] 16 DP-4 DP-6AGTTATGCACAGAGCT [SEQ ID NO: 129] 16 DP-4 SH-2CAGGACGTTGCATAGC [SEQ ID NO: 130] 16 SH-1 SH-3ATGTTGGAGAAGACCTC [SEQ ID NO: 131] 17 SH-1 SH-5GGCCTGATGTACCTATA [SEQ ID NO: 132] 17 SH-4 SH-6GAGCATGCACCTACAC [SEQ ID NO: 133] 16 SH-4 VS-2CTCGGAAATTGCATG [SEQ ID NO: 134] 15 VS-1 VS-3AACTAAGCTCGGCATT [SEQ ID NO: 135] 16 VS-1 VS-5GCAACCTTTAGGGG [SEQ ID NO: 136] 14 VS-4 VS-6CGTCTGCAACTACTACTTC [SEQ ID NO: 137] 19 VS-4 CS-2GTACCGAGGGCAGT [SEQ ID NO: 138] 14 CS-1 CS-3GTAATACATGTTGTGACC [SEQ ID NO: 139] 18 CS-1 CS-5GGCACGGCAAGAAA [SEQ ID NO: 140] 14 CS-4 CS-6GACTTCGCAGACGTAGG [SEQ ID NO: 141] 17 CS-4 PF-2CAAAGTAGTGGTGGG [SEQ ID NO: 142] 15 PF-1 PF-3GATAGTAGTGACAATGCAG [SEQ ID NO: 143] 19 PF-1 PF-5TGCGTGCGTGTATGTA [SEQ ID NO: 144] 16 PF-4 PF-6TGAATGTGCCTTGTGG [SEQ ID NO: 145] 16 PF-4 PE-2AGTAGTAGAAAGCTCAGC [SEQ ID NO: 146] 18 PE-1 PE-3AGACGACCTTCGAGC [SEQ ID NO: 147] 15 PE-1 PA-2TACAGTAGAGAGCTCGG [SEQ ID NO: 148] 17 PA-1 PA-3CAGAGGACCTTAGAAC [SEQ ID NO: 149] 16 PA-1 PA-5GAGCACGACAGGAACG [SEQ ID NO: 150] 16 PA-4 PA-6ACTCCAACGACGCAGA [SEQ ID NO: 151] 16 PA-4

EXAMPLE 3 Effect of the Extent of Biotin Labeling on Capture Efficiency

Tests were conducted to determine the optimal number of biotin labelsper capture sequence probe for TSHC detection. The general TSHC methoddescribed in Example 1 was employed. The capture efficiency of capturesequence probe F15R labeled with one, two, or three biotins, measured bysignal to noise ratio (SIN), were tested. The signal sequence probeemployed was H19. As shown in Table 9, two biotins per capture sequenceprobe were sufficient for optimal capture efficiency. Greater than a 50%increase in SIN was observed using capture sequence probe with twobiotin labels compared to the single biotin labeled capture sequenceprobe. The addition of a third biotin label to the capture sequenceprobe resulted in a decrease in S/N relative to the two-biotin labeledcapture sequence probe.

TABLE 9 EFFECT OF THE EXTENT OF BIOTIN LABELING ON CAPTURE EFFICIENCY #Biotins HSV-1/well RLU CV S/N One 0 54 3% 1.0 One 4.5 × 10{circumflexover ( )}3 236 2% 4.4 One 4.5 × 10{circumflex over ( )}4 1861 3% 34.5One 4.5 × 10{circumflex over ( )}5 15633 7% 289.5 Two 0 46 3% 1.0 Two4.5 × 10{circumflex over ( )}3 296 10%  6.4 Two 4.5 × 10{circumflex over( )}4 2558 1% 55.6 Two 4.5 × 10{circumflex over ( )}5 23369 4% 508.0Three 0 44 22%  1.0 Three 4.5 × 10{circumflex over ( )}3 243 6% 5.5Three 4.5 × 10{circumflex over ( )}4 1820 2% 51.4 Three 4.5 ×10{circumflex over ( )}5 18581 8% 422.3

EXAMPLE 4 Effect of the Distance Between the CSP and the SSP TargetSites on Capture Efficiency

The effect of the distance between capture sequence probe (CSP) andsignal sequence probe (SSP) hybridization sites on a HSV-1 targetnucleic acid on capture efficiency was evaluated. CSPs that hybridize toHSV-1 nucleic acid sequences which are located 0.2 kb, 3 kb, 18 kb, 36kb and 46 kb from the site of SSP hybridization were tested. The generalTSHC method described in Example 1 was employed. The captureefficiencies were 100%, 50%, 30%, 19% and 7%, respectively (Table 10). Asteady decline in relative capture efficiencies was observed as thedistance increased from 0.2 Kb to 46 Kb.

TABLE 10 EFFECT OF DISTANCE BETWEEN TARGET SITES ON CAPTURE EFFICIENCYistance Between Relative Capture CSP SSP Target Site Efficiency BRH19H19 0.2 Kb 100%  F15R H19 3 Kb 50% F6R RH5B 18 Kb 30% F15R RH5B 36 Kb19% F6R H19 46 Kb  7%

EXAMPLE 5 Effect of Fused Capture Sequence Probe on TSHC Detection OfHSV-1

The binding capacity of streptavidin plates was determined to beapproximately 2 pmoles of doubly-biotinylated CSPs per well. Since theCSPs are doubly biotin-labeled, a maximum of 8 CSPs (2 CSPs per SSP) ispreferred in order not to exceed the binding capacity of the wells. Anyincrease in biotin-labeled capture sequence probe above the statedcapacity resulted in a decrease in signal, the so-called “hook effect.”In order to avoid this “hook effect” and still permit the use of greaterthan four SSP-CSP combinations, the effect of synthesizingoligonucleotides that contained the sequences of two CSPs fused together(5′ and 3′ sites) was tested. The fused capture sequence probes mayfunction independently to drive hybridization to the unique targetsites. In another embodiment, the fused probes may bind to two targetsites with the second hybridization favored, since it is essentially auni-molecular reaction with zero order kinetics once the probe hashybridized to the first site. The hybridization may be determined by oneor both mechanisms. Previous experiments showed that two CSPs, VH3, andNC-1, when used together, gave approximately twice the SIN as theindividual CSPs. Unfused capture sequence probes VH-3 and NC-1 were usedat 2.5 pmoles/ml each for a total concentration of 5 pmoles/ml, fusedprobe VH-4 (fusion of VH-3 and NC-1) was used at 2.5 pmole/ml. As shownin Table 11, the fused probe was as effective as the combination of thetwo unfused probes. Therefore, TSHC detection using fused capturesequence probes permits the number of nucleic acid sequences targeted bythe signal sequence probe to be at least doubled without exceeding theplate biotin-binding capacity. The experiment also demonstrates the lackof cross-reactivity of HSV-2 at 10⁷ genomes as shown by the S/N lessthan 2.0.

TABLE 11 COMPARISON OF FUSED VERSUS UNFUSED CAPTURE SEQUENCE PROBES INTSHC DETECTION OF HSV-1 SSP CSP Viral Particles/ml RLU CV S/N RH5B VH-3,NC-1 0 94 14%  1.0 RH5B VH-3, NC-1 10{circumflex over ( )}4 HSV-1 164 5%1.7 RH5B VH-3, NC-1 10{circumflex over ( )}5 HSV-1 1003 4% 10.7 RH5BVH-3, NC-1 10{circumflex over ( )}7 HSV-2 125 6% 1.3 RH5B VH-4 (fused) 097 10%  1.0 RH5B VH-4 (fused) 10{circumflex over ( )}4 HSV-1 181 3% 1.9RH5B VH-4 (fused) 10{circumflex over ( )}5 HSV-1 1070 2% 11.0 RH5B VH-4(fused) 10{circumflex over ( )}7 HSV-2 140 5% 1.4

EXAMPLE 6 Capture Efficiency of Various CSPs and SSPs in TSHC Detectionof HSV-1

The capture efficiency of capture sequence probes (CSPs) for each of thefour HSV-1 specific signal sequence probes (SSPs), H19, RH5B, RH3 andR10, in the detection of HSV-1 by TSHC was evaluated. The criteria usedfor designing the capture sequence probes were: 1) the CSP hybridizationsite is within 1 kb either 5′ or 3′ of the SSP hybridization site on theHSV-1 nucleic acid sequence, preferably within 0.5 kb; and 2) the CSPscontain sequences that are unique to HSV-1, with no stretches ofsequence homology to HSV-2 greater than 10 bases. The CSPs were designedto target the 5′ and 3′ regions adjacent to the SSP hybridization site,preferably with a 5′ CSP and a 3′ CSP for each SSP. The Omiga software(Oxford Molecular Group, Campbell, Calif.) was instrumental in theidentification of such sites. The melting temperature (Tm) of the CSPswas designed to be between 70° C. to 85° C., to conform to the 70° C. to75° C. hybridization temperature used in Hybrid Capture II (HCII) assayfor HSV (Digene). The general TSHC method described in Example 1 wasemployed. Eleven CSPs (which bind to 6 different sites) for H19, sixCSPs (which bind to three unique sites) for RH5B, six CSPs (which bindto six unique sites) for RH3, and two CSPs for R10 were tested. As shownin Table 12, efficient capture sequence probes were found for signalsequence probes H19, RH5B and R10.

TABLE 12 CSPs AND SSPs FOR TSHC DETECTION OF HSV-1 Cap SSP CSP Cap % SSPCSP Cap % SSP CSP % R10 ON-3 100% RH5B TS-1 50% H19 HZ-1 50% R10 ON-3 80% RH5B NC-1 75% H19 HZ-2 20% RH5B VH-4 130%  H19 ZD-1 40% RH5B TS-225% H19 ZD-2 20% RH5B VH-3 50% H19 BRH19 70% H19 VH-2 70% H19 F15R 25%

EXAMPLE 7 Capture Efficiency Of Various CSPs and SSPs in TSHC Detectionof HSV-2

The capture efficiency of capture sequence probes (CSPs) for each of thefour HSV-2 specific signal sequence probes (SSPs), E4A, E4B, Ei8, andi8, in the detection of HSV-2 by TSHC were evaluated. HSV-2 specificcapture sequence probes (CSPs) were designed based on the same criteriaas the HSV-1 CSPs except for the requirement that they be HSV-2specific. Four CSPs for E4A, three CSPs for E4B, and two CSPs each forEi8 and i8 were tested. The general TSHC method described in Example 1was employed. As shown in Table 13, efficient capture sequence probeswere found for i8 and Ei8.

TABLE 13 CSPs AND SSPs FOR TSHC DETECTION OF HSV-2 SSP CSP Cap % i8 NF-1100%  Ei8 NF-2 50% Ei8 LE-3 45%

EXAMPLE 8 Effect of Blocker Probes on HSV-1 and HSV-2 Detection

In an attempt to reduce cross-reactivity of TSHC while allowing thecapture step to take place at room temperature, methods using blockerprobes were developed. Blocker probes comprise sequences that arecomplementary to the capture sequence probes (CSPs) used for detection.These experiments were designed to prevent non-specific hybridization ofthe CSPs to non-targeted nucleic acids present in the sample under thelower stringency conditions, a situation often encountered during theroom temperature capture step.

In one method, blocker probes that are complementary to the full lengthor nearly the full length of the capture sequences probe were used. Theblocker probes were added to the reaction mixture in 10-fold excessrelative to the CSP after hybridization of the CSP and the SSP to thetarget DNA molecule has occurred. Since the blocker probes have similarmelting temperature as the CSPs, the CSPs were hybridized to the targetnucleic acids first to prevent hybridization of the blocker probes tothe CSPs before the hybridization of the CSPs to the target nucleicacids occurred. As shown in Table 14, the addition of the blocker probesresulted in a dramatic reduction in cross-reactivity while these probeshad no effect on the sensitivity of HSV-1 detection. The SIN for thedetection of cross-reactive HSV-2 (10⁷ viral particles/nil) decreasedfrom 5.0 to 0.8 when the blocker probes were used.

In another method, blocker probes that are complementary to only aportion of the CSPs and are shorter than the CSPs were used. The blockerprobes were designed to have melting temperatures above room temperaturebut at least 10° C. below the hybridization temperature of CSPs to thetarget nucleic acids. Since these blocker probes hybridize to the CSPsat temperature below the CSP hybridization temperature to the targetnucleic acids, the blocker probes may be added to the reaction at thesame time as the CSP and SSP without effecting the hybridizationefficiency of the CSPs to the target nucleic acid. These shorter blockerprobes function during the room temperature capture step by hybridizingto the CSPs at the lower temperatures that are encountered during theroom temperature capture step. As shown in Table 15, the addition ofeither single or paired shorter blocker probes in 100-fold excessrelative to the CSPs resulted in a dramatic reduction incross-reactivity but had no effect on sensitivity of HSV-1 detection.The S/N for detecting cross-reactive HSV-2 (10⁷ viral particles/ml)without the blocker probes was 10.6, but was reduced to less than orequal to 1.5 with the addition of the blocker probes.

Therefore, both methods utilizing blocker probes provide a substantialreduction in cross-reactivity. The second method utilizing blockerprobes with lower melting temperature may be preferred because theaddition of blocker probes at the same time as the capture sequenceprobe eliminates the need for an extra step for the detection method.

TABLE 14 EFFECT OF BLOCKER PROBES ADDED POST CAPTURE PROBE HYBRIDIZATIONON TSHC SSP CSP 100x Blocker Probe Viral Particles/ml RLU CV S/N H19HZ-1 None 0 66 7% 1.0 H19 HZ-1 None 10{circumflex over ( )}5 HSV-1 2465% 3.7 H19 HZ-1 None 10{circumflex over ( )}6 HSV-1 1998 2% 30.3 H19HZ-1 None 10{circumflex over ( )}7 HSV-2 327 2% 5.0 H19 HZ-1 ZD-3 0 603% 1.0 H19 HZ-1 ZD-3 10{circumflex over ( )}5 HSV-1 267 4% 4.5 H19 HZ-1ZD-3 10{circumflex over ( )}6 HSV-1 2316 6% 38.6 H19 HZ-1 ZD-310{circumflex over ( )}7 HSV-2 49 2% 0.8

TABLE 15 EFFECT OF BLOCKER PROBES ADDED SIMULTANEOUSLY WITH THE CAPTUREPROBES UPON TSHC DETECTION OF HSV-1 10x SSP CSP Blocker Probe ViralParticle/ml RLU CV S/N H19 HZ-1 none 0 38 15%  1.0 H19 HZ-1 none10{circumflex over ( )}4 HSV-1 71 2% 1.9 H19 HZ-1 none 10{circumflexover ( )}5 HSV-1 389 12%  10.2 H19 HZ-1 none 10{circumflex over ( )}7HSV-2 401 18%  10.6 H19 HZ-1 NG-4 0 39 8% 1.0 H19 HZ-1 NG-410{circumflex over ( )}4 HSV-1 82 5% 2.1 H19 HZ-1 NG-4 10{circumflexover ( )}5 HSV-1 411 18%  10.5 H19 HZ-1 NG-4 10{circumflex over ( )}7HSV-2 57 15%  1.5 H19 HZ-1 EA-1, EA-2 0 37 0% 1.0 H19 HZ-1 EA-1, EA-210{circumflex over ( )}4 HSV-1 75 8% 2.0 H19 HZ-1 EA-1, EA-210{circumflex over ( )}5 HSV-1 419 8% 11.3 H19 HZ-1 EA-1, EA-210{circumflex over ( )}7 HSV-2 49 5% 1.3 H19 HZ-1 NG-7, NG-8 0 42 10% 1.0 H19 HZ-1 NG-7, NG-8 10{circumflex over ( )}4 HSV-1 76 3% 1.8 H19HZ-1 NG-7, NG-8 10{circumflex over ( )}5 HSV-1 471 5% 11.2 H19 HZ-1NG-7, NG-8 10{circumflex over ( )}7 HSV-2 47 9% 1.1

EXAMPLE 9 TSHC Detection Reduces Vector Background

The TSHC assay eliminates the vector contamination problem oftenassociated with the Hybrid Capture II (HC II) detection assay (Digene).As the RNA signal sequence probes used in HC II are generated fromlinearized vector templates, any remaining unlinearized plasmid DNAresults in the production of additional RNA probe sequences specific forvector sequences. In the HC II assay, the RNA/DNA hybrids that form as aresult of these read-through transcripts are captured on the antibodycoated plates and generate signal. In contrast, in the TSHC method, onlythose RNA/DNA hybrids that also hybridize to the capture sequence probesare detected. Accordingly, any detection of vector-related sequences iseliminated. Plasmids SK+, pBR322, DgZ, and 1066 which were known to bedetectable in HSV HC II test (Digene) were tested in the TSHC assayusing two RNA signal sequence probes (H19 and RH5b) and two capturesequence probes (VH-2 and VH-4). Identical sets of RNA probes were thenused in the HC II method and the TSHC method for the detection of HSV-1.The general TSHC method described in Example 1 was employed. As shown inTable 16, while signal to noise ratio in standard HC II ranged from 14to 48, the signal to noise ratio for the TSHC method was less than 2 forall plasmids tested.

TABLE 16 VECTOR BACKGROUND IN TSHC V. HCII DETECTION Method SSP CSPTargets/ml RLU CV S/N TSHC H19 + RH5B VH-2 + 0 94 6% 1.0 VH-4 TSHC H19 +RH5B VH-2 + 4 ng pBS SK+ 137 7% 1.5 VH-4 TSHC H19 + RH5B VH-2 + 2 ngpBR322 99 6% 1.1 VH-4 TSHC H19 + RH5B VH-2 + 4 ng DgX 135 7% 1.4 VH-4TSHC H19 + RH5B VH-2 + 4 ng 1066 107 7% 1.1 VH-4 HC II H19 + RH5B None 094 9% 1.0 HC II H19 + RH5B None 4 ng pBS SK+ 4498 3% 48.1 HC II H19 +RH5B None 2 ng pBR322 1281 8% 13.7 HC II H19 + RH5B None 4 ng DgX 20035% 21.4 HC II H19 + RH5B None 4 ng 1066 1536 2% 16.4

EXAMPLE 10 Sensitivity and Specificity of Detecting HSV-1 and HSV-2 byTSHC

The sensitivity and typing discrimination for the TSHC detection ofHSV-1 and HSV-2 were assessed using the TSHC described in Example 1. Inthe HSV-1 TSHC assay, signal sequence probes H19 and RH5B, capturesequence probes HZ-1, VH-2 and VH-4, and blocker probes NG-7, NG-8,GP-3, GP-4, and GP-1 were used. In the HSV-2 TSHC assay, signal sequenceprobes i8 and Ei8, capture sequence probes NF-1 and NF-2, and blockerprobes HX-4, HX-5 and GP-8 were used. HSV-1 and HSV-2 viral particleswere diluted to various concentrations using the Negative ControlSolution. As shown in FIGS. 4 and 5, while 10⁴ copies of the eitherHSV-1 or HSV-2 (450 copies/well) were detected in the respective assays,there was virtually no detection of the cross-reactive type HSV atconcentrations up to and including 10⁸ copies/ml (4,500,000copies/well). Thus, the HSV-1 and HSV-2 TSHC assays can distinguish thetwo HSV types at a greater than 10,000-fold range of discriminationwhile maintaining excellent sensitivity (450 VP/well).

The HSV-1 TSHC assay shows a linear range of detection ranging from atleast 2×10³ to 5×10³ VP/ml (Table 17). The specificity of the assay isexcellent as no cross-reactivity was detected (S/N is less than or equalto 2) in samples containing HSV-2 at a concentration as high as 2×10⁷ to5×10⁷ viral particles/ml. Similarly, the HSV-2 TSHC assay also showsexcellent specificity, wherein no cross-reactivity was detected insamples containing HSV-1 at a concentration as high as 5×10⁷ viralparticles/mi (Table 18). Similar results were obtained from TSHCdetection of HSV-2 using a dilution series of HSV-2 and HSV-1 viruses(Table 19).

TABLE 17 ANALYTICAL SENSITIVITY AND SPECIFICITY OF THE HSV1 TSHC ASSAYTargets RLU S/N Negative Control 47 1.0 HSV2 @ 5 × 10{circumflex over( )}7 VP/ml 57 1.2 HSV2 @ 2 × 10{circumflex over ( )}7 VP/ml 43 0.9 HSV1@ 5 × 10{circumflex over ( )}3 VP/ml 201 4.3 HSV1 @ 2 × 10{circumflexover ( )}3 VP/ml 107 2.3

TABLE 18 ANALYTICAL SENSITIVITY AND SPECIFICITY OF THE HSV2 TSHC ASSAYTargets RLU S/N Negative Control 40 1.0 HSV1 @ 5 × 10{circumflex over( )}7 VP/ml 78 2.0 HSV1 @ 2 × 10{circumflex over ( )}7 VP/ml 55 1.4 HSV2@ 5 × 10{circumflex over ( )}3 VP/ml 218 5.5 HSV2 @ 2 × 10{circumflexover ( )}3 VP/ml 106 2.7

TABLE 19 DETECTION WITH HSV-2 PROBES USING HSV-1 AND HSV-2 OF DIFFERENTDILUTION Targets RLU S/N Negative Control 43 1.0 HSV1 @ 5 ×10{circumflex over ( )}7 VP/ml 112 2.6 HSV1 @ 2 × 10{circumflex over( )}7 VP/ml 57 1.3 HSV1 @ 1 × 10{circumflex over ( )}7 VP/ml 38 0.9 HSV1@ 1 × 10{circumflex over ( )}6 VP/ml 38 0.9 HSV1 @ 1 × 10{circumflexover ( )}5 VP/ml 33 0.8 HSV1 @ 1 × 10{circumflex over ( )}4 VP/ml 52 1.2HSV1 @ 1 × 10{circumflex over ( )}3 VP/ml 43 1.0 HSV1 @ 1 ×10{circumflex over ( )}2 VP/ml 39 0.9 HSV2 @ 1 × 10{circumflex over( )}7 VP/ml 257173 5980.8 HSV2 @ 1 × 10{circumflex over ( )}6 VP/ml28544 663.8 HSV2 @ 1 × 10{circumflex over ( )}5 VP/ml 3200 74.4 HSV2 @ 1× 10{circumflex over ( )}4 VP/ml 266 6.2 HSV2 @ 5 × 10{circumflex over( )}3 VP/ml 181 4.2 HSV2 @ 1 × 10{circumflex over ( )}3 VP/ml 62 1.4HSV2 @ 1 × 10{circumflex over ( )}2 VP/ml 44 1.0

EXAMPLE 11 Clinical Specimen Testing

A 64-member clinical specimen panel was tested for HSV-1 and HSV-2 usingboth TSHC and HCII methods. The panel included 15 samples containingknown quantities of HSV-1 or HSV-2, and 49 samples known to be negativefor HSV-1 and HSV-2 by PCR testing. Accordingly, the 15 positive sampleswere “Expected” to test positive in both the HCII and TSHC assays, andthe 49 negative samples were “Expected” to test negative in both theHCII and TSHC tests. The general TSHC method described in Example 1 wasemployed. The results using the HCII method and the TSHC method areshown in Tables 20 and 21, respectively. Of the 49 samples “Expected” toyield negative result, 5 samples tested positive and 44 samples testedpositive using the HCII method. In comparison, all 49 samples testednegative using the TSHC method. Therefore, the TSHC method is superiorin specificity to the HCII method in the detection of HSV-1 and HSV-2.

TABLE 20 OBSERVED VERSUS EXPECTED RESULTS FOR HCII DETECTION OF HSV1 ANDHSV2 Expected Result HCII Result Positive Negative Positive 15 5Negative 0 44 Total 15 49

TABLE 21 OBSERVED VS. EXPECTED RESULTS FOR TSHC DETECTION HSV1 AND HSV2Expected Result TSHC Result Positive Negative Positive 14 0 Negative 149 Total 15 49

EXAMPLE 12 Effect of Combining Probes in TSHC Detection of HSV

The effect of combining HSV-1 specific signal sequence probe and capturesequence probe sets on HSV-1 detection was assessed. TSHC detection ofHSV-1 and HSV-2 cross-reactivity was performed separately with twodifferent sets of RNA signal sequence probe /biotinylated capturesequence probe combinations (Set #1: H19 plus HZ-1; and Set #2: RH5bplus the TS-1 and TS-2). TSHC was also performed with both RNA signalsequence probe/biotinylated capture sequence probe sets combined toassess the effect of combining the two probe sets on sensitivity andcross-reactivity. The general TSHC method described in Example 1 wasemployed. The results shown in Table 22 clearly demonstrate an additiveeffect of combining the two probe sets for HSV-1 detection with noapparent increase in HSV-2 cross-reactivity.

TABLE 22 SENSITIVITY IS IMPROVED BY COMBINING HSV-1 SPECIFIC CSPs ANDSSPs Capture Sequence Signal Sequence Probes Probes VP/ml RLU CV S/NHZ-1 H19 0 60 3% 1.0 HZ-1 H19 10{circumflex over ( )}5 HSV-1 267 4% 4.5HZ-1 H19 10{circumflex over ( )}6 HSV-1 2316 6% 38.9 HZ-1 H1910{circumflex over ( )}7 HSV2 49 2% 0.8 TS-1, TS-2 RH5B 0 78 6% 1.0TS-1, TS-2 RH5B 10{circumflex over ( )}5 HSV-1 291 6% 3.8 TS-1, TS-2RH5B 10{circumflex over ( )}6 HSV-1 2368 11%  30.6 TS-1, TS-2 RH5B10{circumflex over ( )}7 HSV2 75 11%  1.0 HZ-1, TS-1, TS-2 H19, RH5B 070 12%  1.0 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}5 HSV-1457 10%  6.5 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}6 HSV-14263 1% 60.9 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}7 HSV2 676% 1.0

EXAMPLE 13 TSHC Detection of HPV18 and HPV45

The relative sensitivity and specificity of TSHC and HCII detection ofHuman Papillomavirus 18 (HPV18) and Human Papillomavirus 45 (HPV45) wascompared. Previous studies have established HPV45 as the mostcross-reactive HPV type to HPV18, and conversely, HPV18 as the mostcross-reactive HPV type to HPV45. In this study, the ability of the twomethods to detect HPV18 and HPV45 was assessed using HPV18 and HPV45plasmid DNA.

Capture sequence probes (CSPs) for each of the four Human Papillomavirustypes: HPV16, HPV18, HPV31, and HPV45, were designed. The criteria usedfor designing the capture sequence probes were: 1) the CSP hybridizationsites do not overlap with the SSP sites; 2) the CSPs contain sequencesunique to one HPV type with no stretches of sequence homology to otherHPV types greater than 12 bases; and 3) the CSPs are of sufficientlength so as to be capable of hybridizing efficiently at 70° C.

The blocker probes for each CSP were designed such that they could beadded simultaneously with the CSP during hybridization to the targetnucleic acid. The blocker probes have a melting temperature of at least37° C. but no higher than 60° C., as calculated by the Oligo 5.0 program(National Biosciences, Inc., Plymouth, Minn.). Two blocker probes wereused for each capture oligonucleotide to maximize the blocker effectduring the room temperature plate capture step. It was also desired thatthe blocker probes for each CSP have similar melting temperatures.

CSPs for each of the HPV types were tested for relative captureefficiency and cross-reactivity to other HPV types. CSPs that providedthe best combination of sensitivity and low cross-reactivity were usedfor the detection of HPV using TSHC.

In TSHC and Hal detection of HPV18, HPV18 DNA was used at aconcentration of 10 pg/ml. HPV45, used for cross-reactivity testing, wasused at 4 ng/ml. The general TSHC method described in Example 1 wasemployed. As shown in Table 23, a signal to noise ratio of 16.9 wasobtained for TSHC detection of HPV18 compared to a ratio of 7.6 obtainedfor HCII detection of HPV18. On the other hand, cross-reactivity withHPV45 was significantly reduced using the TSHC method (S/N of 1.3 forTSHC compared to S/N of 393.3 for HCII). The results clearly show thatcompared to the HCII method, the TSHC method for the detection of HPV18was superior in both sensitivity and specificity. Results obtained inexperiments comparing TSHC and HCII detection of HPV45 demonstrate thatthe TSHC method for the detection of HPV45 is superior in bothsensitivity and specificity (Table 24).

TABLE 23 TSHC DETECTION OF HPV 18 Method Target SSP CSP S/N TSHC 0 18L118-7L 1.0 HPV18 (10 pg/ml) 18L1 18-7L 16.9 HPV45 (4 ng/ml) 18L1 18-7L1.3 HC II 0 18L1 none 1.0 HPV18 (10 pg/ml) 18L1 none 7.6 HPV45 (4 ng/ml)18L1 none 393.3

TABLE 24 TSHC DETECTION OF HPV 45 Method Target SSP CSP S/N TSHC 0 45L1ON-1 1.0 HPV45 (10 pg/ml) 45L1 ON-1 8.4 HPV18 (4 ng/ml) 45L1 ON-1 1.6 HCII 0 45L1 none 1.0 HPV45 (10 pg/ml) 45L1 none 8.2 HPV18 (4 ng/ml) 45L1none 494.0

EXAMPLE 14 Target-Specific Hybrid Capture-Plus Assay Protocol

Hepatitis B Virus (HBV) was used as the model system for the developmentof the target-specific hybrid capture-plus (TSHC-plus) assay for thedetection of target nucleic acids.

The hybridization in the TSHC-plus method (FIG. 6A-6D) may be performedin a single step. In the one-step method, CSPs, SSPs containingpre-hybridized DNA/RNA duplex, bridge probes (FIG. 6B-6D), and blockerprobes are added simultaneously to the target nucleic acids. Ifhybridization is performed in two steps, CSPs, SSPs withoutpre-hybridized DNA/RNA duplex, bridge probes and blocker probes arefirst hybridized to the target nucleic acid. Oligonucleotide probescomplementary to the single stranded nucleic acid sequence in the SSPare then added to the reaction to form the DNA/RNA duplexes. The hybridsare then detected using anti-RNA/DNA antibody as described in Example 1.

Experiments were carried out to detect HBV using TSHC-plus (Examples15-18). The method shown in FIG. 6A was used. Human hepatitis B virus(HBV adw2) plasmid DNA of known concentration (Digene Corp) was dilutedusing HBV negative Sample Diluent (Digene). Various dilutions were madeand aliquoted into individual tubes. The negative Sample Diluent wasused as a negative control. A half volume of the Denaturation Reagent5100-0431 (Digene) was added to the test samples. Test samples wereincubated at 65° C. for 45 minutes to denature the nucleic acids in thesamples.

Following denaturation of the HBV sample, a hybridization solutioncontaining capture sequence probes (CSPs), blocker probes, signalsequence probe comprising a M13 DNA/M13 RNA duplex, and a bridge probeof a single-stranded or partially single stranded DNA sequence capableof hybridizing to both an SSP and HBV sequences was added to thesamples, and incubated at 65° C. for 1-2 hours. Alternatively, thedenatured samples were incubated for 1 hour with a hybridizationsolution containing capture sequence probes (CSPs), blocker probes andM13 DNA plasmid containing HBV complementary sequences for 1 hour.Following the incubation, M13 RNA was added to the reaction and theincubation was continued for an additional hour at 65° C.

Tubes containing reaction mixtures were cooled at room temperature for 5minutes and aliquots were taken from each tube and transferred toindividual wells of a 96-well streptavidin plate (Digene). The plateswere shaken at 1100 rpms for 1 hour at room temperature. The solutionwas then decanted and the plates were washed four times with SNM washbuffer (Digene). The alkaline-phosphatase anti-RNA/DNA antibody DR-I(Digene) was added to each well and incubated for 30 minutes at roomtemperature. The DR-I (Digene) was then decanted and the plates werewashed four times with SNM wash buffer (Digene). Following removal ofthe residual wash buffer, luminescent substrate (CDP-Star, Tropix Inc.)was added to each well and incubated for 15 minutes at room temperature.Individual wells were read on a plate luminometer to obtain relativelight unit (RLU) signals.

EXAMPLE 15

The following tables describe the various probes tested in theexperiments described in Examples 16-18.

TABLE 25 CAPTURE SEQUENCE PROBES FOR HBV Size Location within ProbeSequence (bp) HBV Strand HBV C1 GCTGGATGTGTCTGCGGCGTTTTATCAT 28 374-401Sense (SEQ ID NO: 152) HBV C2 ACTGTTCAAGCCTCCAAGCTGCGCCTT 27 1861-1877Sense (SEQ ID NO: 153) HBV C3 ATGATAAAACGCCGCAGACACATCCAGCG 32 370-401Anti- ATA (SEQ ID NO: 154) sense

TABLE 26 HBV/M13 CLONES FROM WHICH SSPs ARE PREPARED Insert SizeLocation Clone name Vector Cloning site (bp) within HBV SA1 M13 mp 18Eco RI, Hind III 35 194-228 SA2 M13 mp 18 Eco RI, Hind III 34 249-282SA1a M13 mp 19 Eco RI, Hind III 35 194-228 SA2a M13 mp 19 Eco RI, HindIII 34 249-282 SA4 M13 mp 19 Eco RI, Hind III 87 1521-1607

TABLE 27 HBV BLOCKER PROBES Size CSP to which Probe Sequence (bp)it hybridizes B1 ATGATAAAACGCCG (SEQ ID NO: 155) 14 HBV C1 B2CAGACACATCCAGC (SEQ ID NO: 156) 14 HBV C1 B3AAGGCACAGCTTG (SEQ ID NO: 157) 13 HBV C2 B4GAGGCTTGAACAGT (SEQ ID NO: 158) 14 HBV C2 B5TATCGCTGGATGTGTC (SEQ ID NO: 159) 16 HBV C3 B6TCGGCGTTTTATCATG (SEQ ID NO: 160) 16 HBV C3

EXAMPLE 16 Effect of Blocker Probes on TSHC-Plus Detection of HBV

During room temperature capture step, excess SSP (M13 RNA/HBV-M13 DNAduplex) non-specifically hybridizing to the CSP are immobilized onto theplate which results in high background signals. In an attempt to reducebackground signal, blocker probes were employed in TSHC-Plus detectionof HBV. The blocker probes were designed to be much shorter than theCSPs so that they are only capable of hybridizing to the capture probesat temperatures well below the hybridization temperatures used in theassay.

Blocker probe sets consisting of two separate oligonucleotides that arecomplementary to the CSPs were used. The blocker probes were added tothe hybridization mixture in 10-fold excess relative to the CSPs. Sincethe blocker probes are much shorter than the CSPs, they do not hybridizewith CSPs at the target hybridization temperature and therefore do notinterfere with the hybridization of the CSPs to the target nucleicacids. Following the hybridization of CSP and target nucleic acids, thesamples were subjected to a room temperature capture step during whichthe blocker probes hybridize with excess CSPs, thus preventing them fromhybridizing to the SSPs. As shown in Table 28, the use of the blockerprobes in the hybridization reaction greatly reduced the backgroundsignals of the assay.

TABLE 28 EFFECT OF BLOCKER PROBES ON HBV DETECTION Capture Probe Blockerprobe Background Signal (RLU) HBV C1 no 17892 HBV C1 B1, B2 424 HBV C2no 9244 HBV C2 B3, B4 398

EXAMPLE 17 Effect of the Length of SSP on TSHC-Plus Detection of HBV

The effect of the length of the DNA sequence inserted into the M13vector for generating the SSP on TSCH-Plus detection of HBV was studied.A positive control containing 20 μg/ml of HBV plasmid DNA was used. Asshown in Table 29, the use of a longer HBV complementary sequence in theSSP (87 base pairs) resulted in a substantial increase in signal ofdetection. The effect is unlikely due to sub-optimal hybridizationtemperature condition since the Tm of the shorter probes is 15 degreeabove the hybridization temperature. As the M13 RNA/DNA duplex formed inthe SSP may act to partially block the complementary DNA sequence in theprobe from hybridizing to the HBV sequences in the target nucleic acids,longer complementary sequences in the SSP may overcome this block.

TABLE 29 EFFECT OF THE LENGTH OF THE COMPLEMENTARY SEQUENCE IN THE SSPON TSHC-PLUS DETECTION OF HBV Size of the HBV Tm of the HBV Hybridi-Target DNA Sequence Target DNA Se- zation Signal SSP in SSP (bp) quencein SSP temperature (RLU) SA1 35 83° C. 65° C. 1741 SA2 34 80° C. 65° C.1857 SA4 87 108° C.  65° C. 7978

EXAMPLE 18 TSHC-Plus and HC II DETECTION of HBV

The relative sensitivity of TSHC-Plus and HC II (Hybrid Capture II,Digene) detection of HBV was compared. HBV positive standards of threedifferent concentrations were tested in the experiments. As shown inTable 30, the signals obtained using the TSHC-Plus detection method wereapproximately two-fold higher than those obtained using the HC IIdetection method.

TABLE 30 TSHC-PLUS AND HC II DETECTION OF HBV* Target HBV ConcentrationMethod Control 10 pg/ml 20 pg/ml 100 pg/ml HC II 48 2355 4225 21438 TSHCPlus 285 4856 7978 37689 *Signal measured as relative light unit (RLU)

EXAMPLE 19 Sample Preparation for Target Specific Hybrid CaptureDetection of SNPs

An embodiment of the TSHC method for detecting SNPs provides the HybridCapture-SNP (HC-SNP) method that is demonstrated herein using p53 DNA asthe target molecule and discriminating polymorphisms or SNPs at codon 72of the p53 coding region (Kawajiri, et al. Carcinogenesis. 14:1085-1089,1993). The two p53 polymorphisms on the anti-sense strand at codon 72,are gCg, which encodes Arginine (Arg), and the p53 codon 72, on theanti-sense strand, gGg, that encodes Proline (Pro). The twopolymorphisms are referred to as p53Arg and p53Pro. This is a SNP wherethe HC-SNP method is used for specific detection of the nucleotide. Itis understood that the HC-SNP method is not limited to these specifictypes of probes, probe labels, and targets, but can also encompass thefull scope of variations described for the TSHC method.

Samples comprising either PCR amplicons or genomic DNA were used as atarget for polymorphism detection in the HC-SNP embodiment. Usinggenomic DNA may be particularly beneficial for diagnostic applications.For the preparation of PCR amplicons, two primers were used, forexample, the Upper Primer—5′-AAGACCCAGGTCCAGATGAAG-3′ (SEQ ID NO:161)and the Lower Primer—5′-AGAATGCAAGAAGCCCAGAC-3′ (SEQ ID NO:162)(described by Klaes et al., J. Mol. Med. 77:299-302, 1999). Theseprimers were specifically chosen for amplification of a p53 exon 4region (182 base pairs), utilizing a program comprising: a) 95° C. for 4minutes; b) 94° C. for 40 seconds; c) 62° C. for 40 seconds; d) 72° C.for 40 seconds; e) 72° C. for 6 minutes; and f) 4° C. for storage orprior to use, wherein steps b-d are repeated for 25 to 45 cyclesdepending on the quality of DNA template. PCR amplicons were thendiluted to 1:1000 or 1:100 in TE (10 mM Tris; 1 mM EDTA), pH7.4, priorto testing. Non-limiting examples of genomic DNA samples for thepreparation of genomic DNA include, but are not limited to, humanfluids, cells, tissues, and archival tissues in paraffin blocks. GenomicDNA isolation was performed using the appropriate kits (Qiagen).Approximately, 10-20 μg of isolated genomic DNA per test pair wasrequired for direct polymorphism detecting bypassing the targetamplification step.

Each DNA target was tested with p53-Arg specific and p53-Pro specificcapture oligos separately. Signal to noise (S/N) ratios were calculated,and the ratio of p53-Arg specific S/N over p53-Pro specific S/N wereused to identify the sample genotype. An example of the SNP test resultsfor determining the homozygotes (Arg/Arg or Pro/Pro) versusheterozygotes (Arg/Pro) are shown in Table 31. The results of thesetests were confirmed by Wave analysis (Transgenomic; Santa Clara,Calif.) and DNA sequence analysis.

EXAMPLE 20 Target Specific Hybrid Capture Method for Detecting SNPs

Plasmid DNA (p53-Arg and p53-Pro) was prepared from bacterial culturesusing Qiaprep Spin Miniprep Kit (Qiagen, Inc.; Valencia, Calif.).Genomic DNA (HeLa, SiHa, and Jurkat) was prepared from the cell linesusing DNeasy Tissue Kit (Qiagen, Inc.). Plasmid DNA and clinical sampleDNA were amplified using the PCR method previously described (45cycles). Prior to use, PCR amplified DNA was diluted 1:1000 in TE, pH7.4, and plasmid DNA samples were diluted to 100 pg/ml in TE, pH 7.4.Five microliters of diluted PCR amplified or plasmid DNA was used pertest. Fifty microliters of extracted genomic DNA samples were used pertest containing 5 μg, 7 μg, and 10 μg for HeLa, Jurkat, and SiHa,genomic DNA respectively. Each sample was tested twice independently foreach assay. The first test was performed using the p53-Arg CSP and p53SSP. The second test was performed using the p53-Pro CSP and p53 SSP.

A mixture of water and DNA target at a final volume of 50 μl per well,was added to the hybridization microplate. Denaturation Reagent5100-0431 (Digene) (25 μl) was added per well. The plate was coveredwith a plate sealer and agitated for 10-30 seconds at 1100 rpm on aplate shaker. The reactions were denatured at 65° C. for 25 minutes inthe microplate heater I (Robbins Dcientific Corp.; Sunnyvale, Calif.).During the denaturation step, the probe mixtures were prepared. Thep53-Arg specific probe mixture consisted of 15 pmoles/ml of 16-base longArg-specific CSP, 600 ng/ml of p53 SSP, and 4× Probe Diluent (Digene).The p53-Pro specific probe mixture consisted of 15 pmoles/ml of 16-baselong Pro-specific CSP, 600 ng/ml of p53 SSP, and 4× Probe Diluent(Digene). Each probe mixture (25 μl each) was added to the denaturedsample. The plate was covered with a plate sealer and agitated for 10-30seconds at 1100 rpm using a plate shaker. The samples were allowed tohybridize at 65° C. for 1 hour in the microplate heater. Hybridizedsamples were incubated at room temperature for 5-10 minutes (to decreasethe temperature of the plate). Hybridization reactions were transferredto a 96-well streptavidin (SA) plate (Digene), and covered with a platesealer. The hybrids were captured onto the SA plate at 45° C. for 45minutes with agitation at 900 rpm. Immobilization of CSP hybridizedtargets can be performed in hybridization solution placed into wells ofa 96-well plate, for example, and the plate is shaken for 15 minutes to2 hours at temperatures ranging from 20° C. to 90° C., preferably atroom temperature for 1 hour shaking at 1100 rpms. Capture temperaturesabove room temperature may be preferred for added levels of stringencyas hybridization (and “promiscuous hybridization”) does occur during theplate capture step. Supernatant was decanted and 100 μl per well of DR-1(Digene) was added for detection of captured RNA/DNA hybrids. The platewas incubated at room temperature for 30 minutes without agitation.Supernatant was discarded and the plate was washed twice with roomtemperature Sharp Wash Buffer. The wells were then re-filled with SharpWash Buffer and the plate was incubated at 60° C. for 10 minutes. Theplate was then washed twice with room temperature Sharp Wash Buffer, andonce with room temperature Hybrid Capture 2 Wash Buffer. The plate wasblotted from residual wash buffer (using kimtowels). A chemiluminescentphosphorylated substrate, DR-2 (100 μl/well) was added and reactionswere incubated at room temperature for 15 minutes without agitation. Theactivated substrate was measured and analyzed using a plate luminometer(See Table 31).

TABLE 31 GENOTYPE DATA FROM HC-SNP S/N using S/N using P53 DNAArg-specIfic Pro-specific Arg/Pro TARGET capture oligo capture oligoRatio Genotype P53-Arg DNA, 98.9 4.5 21.91 Arg 100 pg/ml homozygousP53-Pro DNA, 10.2 68.0 0.15 Pro 100 pg/ml homozygous P53-Arg/Pro 56.454.1 1.04 Arg/Pro DNA, 100 pg/ml heterozygous P53-Arg PCR 1350.1 7.9170.90 Arg homozygous P53-Pro PCR 88.0 1093.8 0.08 Pro homozygousP53-Arg/Pro 874.3 506.5 1.73 Arg/Pro PCR heterozygous HeLa DNA, 5 μg10.8 7.0 1.54 Arg/Pro per well heterozygous SiHa DNA, 10 3.8 15.5 0.25Pro μg per well homozygous Jurkat DNA, 7 23.2 1.6 14.5 Arg μg per wellhomozygous PCR Clinical 162.6 106.2 1.53 Arg/Pro Sample 1 heterozygousPCR Clinical 51.9 652.5 0.08 Pro Sample 2 homozygous PCR Clinical 345.32.3 150.13 Arg Sample 3 homozygous

The above description of various preferred embodiments has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or limiting to the precise forms disclosed.Obvious modifications or variations are possible in light of the aboveteachings. The embodiments discussed were chosen and described toprovide illustrations and its practical application to thereby enableone of ordinary skill in the art to utilize the various embodiments andwith various modifications as are suited to the particular usecontemplated. All such modifications and variations are within thesystem as determined by the appended claims when interpreted inaccordance with the breadth to which they are fairly, legally andequitably entitled.

1. A nucleic acid probe having a nucleic acid sequence consisting of asequence selected from the group consisting of SEQ ID NO: 1 through SEQID NO:
 160. 2. A nucleic acid probe set comprising: a) at least onecapture sequence probe, wherein the capture sequence probe is 15 to 100nucleotides in length and comprises at least one sequence that is atleast 75% complementary to a first target region of a target nucleicacid, wherein the first target region is at least 6 nucleotides inlength; b) at least one signal sequence probe; c) optionally, at leastone bridge probe; and d) optionally, at least one blocker probe at least5 nucleotides in length, wherein the blocker probe is at least 75%complementary to at least a portion of the capture sequence probe,wherein either the signal sequence probe or the bridge probe is capableof hybridizing under stringent hybridization conditions to a secondtarget region of the target nucleic acid, wherein the first targetregion and the second target region do not overlap.
 3. The nucleic acidprobe set of claim 2, wherein the first target sequence is from 20 to 40nucleotides in length.
 4. The nucleic acid probe set of claim 2, whereinthe target nucleic acid is HSV1 and the capture sequence probe comprisesat least one sequence selected from the group consisting of SEQ ID NO: 1to SEQ ID NO:
 29. 5. The nucleic acid probe set of claim 4 comprising atleast one blocker probe, wherein the blocker probe comprises a sequenceselected from the group consisting of SEQ ID NO: 30 to SEQ ID NO:
 49. 6.The nucleic acid probe set of claim 2, wherein the target nucleic acidis HSV2 and the capture sequence probe comprises at least one sequenceselected from the group consisting of SEQ ID NO: 50 to SEQ ID NO:
 62. 7.The nucleic acid probe set of claim 6 comprising at least one blockerprobe, wherein the blocker probe comprises a sequence selected from thegroup consisting of SEQ ID NO: 63 to SEQ ID NO:
 74. 8. The nucleic acidprobe set of claim 2, wherein the target nucleic acid is an HPV16nucleic acid and the capture sequence probe comprises at least onesequence selected from the group consisting of SEQ ID NO: 75 to SEQ IDNO:
 78. 9. The nucleic acid probe set of claim 8 comprising at least oneblocker probe, wherein the blocker probe comprises a sequence selectedfrom the group consisting of SEQ ID NO: 122 to SEQ ID NO:
 129. 10. Thenucleic acid probe set of claim 2, wherein the target nucleic acid is anHPV31 nucleic acid and the capture sequence probe comprises at least onesequence selected from the group consisting of SEQ ID NO: 79 to SEQ IDNO:
 82. 11. The nucleic acid probe set of claim 10 comprising at leastone blocker probe, wherein the blocker probe comprises a sequenceselected from the group consisting of SEQ ID NO: 130 to SEQ ID NO: 137.12. The nucleic acid probe set of claim 2, wherein the target nucleicacid is an HPV18 nucleic acid and the capture sequence probe comprisesat least one sequence selected from the group consisting of SEQ ID NO:83 to SEQ ID NO:
 95. 13. The nucleic acid probe set of claim 12comprising at least one blocker probe, wherein the blocker probecomprises a sequence selected from the group consisting of SEQ ID NO:112 to SEQ ID NO: 117, SEQ ID NO: 147, SEQ ID NO: 150, and SEQ ID NO:151.
 14. The nucleic acid probe set of claim 2, wherein the targetnucleic acid is an HPV45 nucleic acid and the capture sequence probecomprises at least one sequence selected from the group consisting ofSEQ ID NO: 96 to SEQ ID NO:
 109. 15. The nucleic acid probe set of claim12 comprising at least one blocker probe, wherein the blocker probecomprises a sequence selected from the group consisting of SEQ ID NO:110 to SEQ ID NO: 111, SEQ ID NO: 118 to SEQ ID NO: 121, and SEQ ID NO:138 to SEQ ID NO:
 149. 16. The probe set of claim 2, wherein the capturesequence probe comprises complementary sequences to two or more distinctregions of the target nucleic acid.
 17. The probe set of claim 2,wherein the signal sequence probe is capable of hybridizing understringent hybridization conditions to the second target region of thetarget nucleic acid.
 18. The probe set of claim 2 comprising a bridgeprobe, wherein the bridge probe is capable of hybridizing understringent hybridization conditions to the second target region of thetarget nucleic acid, and wherein the signal sequence probe is capable ofhybridizing under stringent hybridization conditions to the bridgeprobe, but not to the target nucleic acid.
 19. The probe set of claim 2,wherein the blocker probe has a melting temperature at least 10 degreeslower than that of the capture sequence probe.
 20. The probe set ofclaim 2, wherein the first target region and the second target regionare within 50,000 bases of each other.
 21. The probe set of claim 20,wherein the distance between first target region and the second targetregion is less than 3,000 bases.
 22. The probe set of claim 20, whereinthe distance between first target region and the second target region isless than 1,000 bases.
 23. The probe set of claim 2, wherein the capturesequence probe comprises at least one sequence that is 100%complementary to a first target region of a target nucleic acid.
 24. Theprobe set of claim 2, wherein the capture sequence probe contains one ormore modifications in the nucleic acid which allows specific capture ofthe probe onto a solid phase.
 25. The probe set of claim 2, wherein thesignal sequence probe is unlabeled.